Comparison of methods for recovery of Escherichia coli and Staphylococcus aureus from seeded laundry fabrics

To assess the effect of laundry procedures on fabric-associated bacteria, a standard method of enumeration is needed. We evaluated six methods for enumeration of Escherichia coli and Staphylococcus aureus seeded (10(2) and 10(5) CFU/100 cm2 of fabric area) onto sterilized hospital sheets and terry . Two methods involved maceration of seeded swatches in broth followed by passage of the broth through a 0.45-micron-pore-size, 47-mm-diameter filter membrane. Three methods involved agitation of seeded swatches in broth with a paint shaker and membrane filtration of the broth to recover eluted bacterial cells, and the final method involved direct enumeration of cells on fabrics by overlaying seeded swatches with agar containing triphenyltetrazolium chloride as an indicator. The most convenient recovery method employed a 90-s agitation followed by serial dilution of broths and membrane filtration. This method provided 44/57% (low seed/high seed) recovery of E. coli from sheets and 133/31% from terry and 34/74% recovery of S. aureus from sheets and 58/57% from terry . Although maceration provided similar recovery of E. coli and S. aureus, it is a less-practical method. The direct enumeration method was ineffective for enumerating gram-positive bacteria. We conclude that either the agitation or maceration method used enumerated the seeded bacteria to within 1 log10 of their expected number and can be used to assess the bactericidal effectiveness of various steps in the laundering process.     

Identification of Staphylococcus aureus and Escherichia coli isolated from Egyptian food by conventional and molecular methods

Staphylococcus aureus and Escherichia coli were enumerated and isolated from ready-to-eat vegetables salad and meat luncheon on their selective media (Baird-parker and Macconkey agar, respectively). Twenty suspicious colonies of each (10 from each product) were randomly chosen and identified using conventional based on morphological and physiological characteristics. S. aureus and E. coli isolates which gave the highest pathogenicity were chosen for identification and confirmation with molecular method based on 16S rRNA gene. The PCR amplification method of 16S rRNA gave the same identification results as conventional method, but it was sensitive and fast. This molecular method takes about 48 h in comparison with 6 days for conventional method. The 16S rRNA of S. aureus and E. coli were deposited in the Genebank database under accessions (AB599719.1 and AB599716.1, respectively).     

Regulation of glucosamine utilization in Staphylococcus aureus and Escherichia coli.

Glucosamine- or N-acetylglucosamine-requiring mutants of Staphylococcus aureus 209P and Escherichia coli K12, which lack glucosamine-6-phosphate synthetase [2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (amino-transferring); EC 5.3.1.19], were isolated. Growth of these mutants on glucosamine was inhibited by glucose, but growth on N-acetylglucosamine was not. Addition of glucose to mutant cultures growing exponentially on glucosamine inhibited growth and caused death of bacteria, though chloramphenicol prevented death. Uptake of glucosamine by S. aureus and E. coli mutants was severely inhibited by glucose whereas uptake of N-acetylglucosamine was only slightly inhibited. Uptake of glucose was not inhibited by either glucosamine or N-acetylglucosamine. In glucosamine auxotrophs, glucose causes glucosamine deficiency which interrupts cell wall synthesis and results in some loss of viability in the presence of continued protein synthesis.     

Transduction of plasmid determinants in Staphylococcus aureus and Escherichia coli.

Buoyant density analysis of transducing lysates derived from Staphylococcus aureus and Escherichia coli indicated that phage particles bearing plasmid determinants contain a quantity of DNA equivalent to that found in the lytic particles. Transducing particles that bear plasmid determinants smaller than viral DNA must therefore contain a quantity of DNA in excess of a single plasmid genome. In the E. coli P1vir system, a dependence upon host-mediated recombination for the transduction of small plasmids, but not for large R factors or chromosomal genes, was observed. However, no evidence for the involvement of such functions in the transduction of S. aureus plasmids was obtained. Although the origin of the additional DNA in plasmid transducing particles has not been identified, circumstantial evidence has been presented in the staphylococcal system indicating that transducing particles carrying a small tetracycline plasmid are not formed by the wrapping of multiple copies of this plasmid DNA.     

Nisin stimulates oxygen consumption by Staphylococcus aureus and Escherichia coli.

Nisin stimulated oxygen consumption by nongrowing, glucose-metabolizing Staphylococcus aureus and Escherichia coli cells, indicating a protonophore mode of action. A similar stimulation in E. coli cells osmotically stressed to disrupt the outer cell membrane confirmed the cytoplasmic membrane as the site of nisin action and showed that nisin uptake was not prevented by the outer membrane.     

Expression of a clone specific DNA sequence from Staphylococcus aureus in Escherichia coli.

Staphylococcus aureus produces a large number of factors thought to contribute to virulence, although the precise role of some of these individual factors is not clearly defined. To investigate whether specific virulence factors might be responsible for the selection and dominance of certain genotypes of methicillin- and multiply resistant S. aureus (MRSA), the method of subtractive hybridisation was used to identify conserved DNA sequences associated with the clinical, clonal populations of S. aureus. The findings described in this report indicate that the method of subtractive hybridisation is a valuable tool to identify clone specific virulence factors, which might be of potential as diagnostic markers and as alternative vaccine targets.     

Characterization of the glutamyl endopeptidase from Staphylococcus aureus expressed in Escherichia coli

V8 protease, a member of the glutamyl endopeptidase I family, of Staphylococcus aureus V8 strain (GluV8) is widely used for proteome analysis because of its unique substrate specificity and resistance to detergents. In this study, an Escherichia coli expression system for GluV8, as well as its homologue from Staphylococcus epidermidis (GluSE), was developed, and the roles of the prosegments and two specific amino acid residues, Val69 and Ser237, were investigated. C-terminal His(6)-tagged proGluSE was successfully expressed from the full-length sequence as a soluble form. By contrast, GluV8 was poorly expressed by the system as a result of autodegradation; however, it was efficiently obtained by swapping its preprosegment with that of GluSE, or by the substitution of four residues in the GluV8 prosequence with those of GluSE. The purified proGluV8 was converted to the mature form in vitro by thermolysin treatment. The prosegment was essential for the suppression of proteolytic activity, as well as for the correct folding of GluV8, indicating its role as an intramolecular chaperone. Furthermore, the four amino acid residues at the C-terminus of the prosegment were sufficient for both of these roles. In vitro mutagenesis revealed that Ser237 was essential for proteolytic activity, and that Val69 was indispensable for the precise cleavage by thermolysin and was involved in the proteolytic reaction itself. This is the first study to express quantitatively GluV8 in E. coli, and to demonstrate explicitly the intramolecular chaperone activity of the prosegment of glutamyl endopeptidase I.     

Magnitude of the protonmotive force in respiring Staphylococcus aureus and Escherichia coli.

The membrane potential and pH gradient developed across the plasma membranes of whole cells of Staphylococcus aureus and spheroplasts of Escherichia coli were estimated. The distributions of potassium ions in the presence of valinomycin and the pH gradient across the membrane were determined from the changes in pK and pH observed in the external medium during transition from the energized respiring state to the de-engerized resting condition. The protonmotive force in respiring cells was estimated at 211 mV for S. aureus and 230 mV for E. coli at external pH values of approximately 6.5. The adequacy of these protonmotive forces as a driving force for substrate accumulation or adenosine 5'-triphosphate synthesis is discussed.     

Sensitivity and resistance of Escherichia coli and Staphylococcus aureus to chlorhexidine

Escherichia coli PC1349 and Staphylococcus aureus 6571 were sensitive to low concentrations of chlorhexidine diacetate, as determined by minimal inhibitory concentration tests. Lack of bactericidal response to 30 μ g/ml was due to the fact that adsorption of biocide to the cells was very slight in suspensions of high cell density and was not due to emerging resistance. Attempts by various methods to induce stable resistance in these organisms failed, despite reports that resistant strains have been isolated.     

Cloning and sequence determination of six Staphylococcus aureus beta-lactamases and their expression in Escherichia coli and Staphylococcus aureus

The plasmid-encoded beta-lactamase genes of six strains of Staphylococcus aureus were cloned and shown to be expressed in Escherichia coli. The cloned genes were re-introduced into S. aureus via a shuttle vector, and expressed beta-lactamase. However, clones containing only the small amount of DNA found necessary for expression of ampicillin resistance in E. coli did not express beta-lactamase in S. aureus. Much larger pieces of DNA from the original plasmid were necessary to obtain expression in S. aureus. Some of the six strains of S. aureus synthesized beta-lactamase constitutively and some released only a small proportion of the enzyme into the medium. Both these characteristics were maintained in the clones so it is concluded that they are features either of the gene itself or of the surrounding DNA. The cloned genes were sequenced and the putative amino acid sequences of the beta-lactamases were compared. There are several differences between the sequences and in particular one change in the N-terminal region, at a position believed to be especially important for export of proteins from the cell, is thought to have a key effect on whether or not the enzyme is found in the medium.     

Nucleotide sequence and expression of the beta-lactamase gene from Staphylococcus aureus plasmid pI258 in Escherichia coli, Bacillus subtilis, and Staphylococcus aureus.

The structural gene for beta-lactamase in the Staphylococcus aureus plasmid pI258 was cloned into a Staphylococcus aureus-Bacillus subtilis-Escherichia coli shuttle vector, pWN101, and the nucleotide sequence of the gene was determined. pWN101 was structurally stable and the beta-lactamase gene was expressed efficiently from its native promoter and ribosome-binding site in all three hosts.     

The phototoxicity of phenothiazinium derivatives against Escherichia coli and Staphylococcus aureus

Phenothiazinium dyes, and derivatives, were tested for toxicity to Escherichia coli and Staphylococcus aureus. The dyes were generally lipophilic (log P>1) and showed inherent dark toxicity (minimum lethal concentrations: 3.1-1000 microM). Dye illumination (total light dose of 3.15 J cm(-1) over 30 min) led to up to eight-fold reductions in minimum lethal concentrations. Most of the illuminated dyes showed significant relative singlet oxygen yields (phi'delta: 0.18-1.35) suggesting a type II mechanism of generating a phototoxic response. Although generally up to six-fold more effective against S. aureus, the dyes tested efficiently killed E. coli and may be of particular use in combating Gram-negative pathogens.     

Cloning and expression of the Staphylococcus aureus glucosaminidase in Escherichia coli

Abstract A fragment of Staphylococcus aureus DNA encoding the glucosaminidase determinant was cloned in Escherichia coli by inserting the Sau 3A genomic fragments in the Bam HI site of the plasmid vector pBR322. One clone selected on the basis of its lytic activity was shown to contain a hybrid plasmid (pEU213) carrying a 4.7 kb insert of S. aureus DNA. Lytic activity was tested using different assays, and the enzyme production was confirmed by immunological reactions. An appreciable reduction of lytic activity was noted after few subcultures. The E. coli carrying pEU213 had a slower growth rate and increased autolytic activity compared to the parental strain. The possible reasons for this behavior are discussed.     

Cloning and expression of the Staphylococcus aureus protein A gene in Escherichia coli.

A gene bank of Staphylococcus aureus strain Cowan I was established using an E. coli HB101/pBR327 host-vector system. Recombinants expressing staphylococcal protein A (SPA) were detected using an IgG-binding assay. A 3.2 Kb DNA fragment directing the synthesis of SPA in E. coli was identified. SPA produced by E. coli was characterised in minicells and by Western blotting and double diffusion experiments.     

Evaluation of culture media for recovery of Staphylococcus aureus from swimming pools.

Several selective media were evaluated for the primary isolation and enumeration of Staphylococcus aureus from halogenated indoor swimming pool waters. Standard plate counts of the viable population and total coliform densities were also determined to ascertain their value as indicator systems. All studies were done with membrane filters. The most selective, accurate, and reliable medium was Vogel-Johnson (VJ) medium supplemented with 0.5% pyruvate. This medium recovered two times more typical colonies than VJ medium alone, and subsequent identification of these well-defined black colonies proved that approximately 80% were S. aureus. The S. aureus recoveries correlated well with halogen levels and bather density use also. In contrast, VJ medium alone was 60% selective for S. aureus, and VJ medium supplemented with catalase did not increase either the percent recovery or the selectivity over that of VJ medium alone. Standard plate counts did not correlate with halogen levels, bather density, or total viable colonies. Coliforms were rarely recovered from indoor pool waters and were not considered to be useful indicators of water quality.